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Replacing uridine with 2-thiouridine enhances the rate and fidelity of nonenzymatic RNA primer extension.

ABSTRACT: The nonenzymatic replication of RNA oligonucleotides is thought to have played a key role in the origin of life prior to the evolution of ribozyme-catalyzed RNA replication. Although the copying of oligo-C templates by 2-methylimidazole-activated G monomers can be quite efficient, the copying of mixed sequence templates, especially those containing A and U, is particularly slow and error-prone. The greater thermodynamic stability of the 2-thio-U(s(2)U):A base pair, relative to the canonical U:A base pair, suggests that replacing U with s(2)U might enhance the rate and fidelity of the nonenzymatic copying of RNA templates. Here we report that this single atom substitution in the activated monomer improves both the kinetics and the fidelity of nonenzymatic primer extension on mixed-sequence RNA templates. In addition, the mean lengths of primer extension products obtained with s(2)U is greater than those obtained with U, augmenting the potential for nonenzymatic replication of heritable function-rich sequences. We suggest that noncanonical nucleotides such as s(2)U may have played a role during the infancy of the RNA world by facilitating the nonenzymatic replication of genomic RNA oligonucleotides.

SUBMITTER: Heuberger BD

PROVIDER: S-EPMC4985000 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Replacing uridine with 2-thiouridine enhances the rate and fidelity of nonenzymatic RNA primer extension.

Heuberger Benjamin D BD Pal Ayan A Del Frate Francesca F Topkar Ved V VV Szostak Jack W JW

Journal of the American Chemical Society 20150216 7

The nonenzymatic replication of RNA oligonucleotides is thought to have played a key role in the origin of life prior to the evolution of ribozyme-catalyzed RNA replication. Although the copying of oligo-C templates by 2-methylimidazole-activated G monomers can be quite efficient, the copying of mixed sequence templates, especially those containing A and U, is particularly slow and error-prone. The greater thermodynamic stability of the 2-thio-U(s(2)U):A base pair, relative to the canonical U:A ...[more]

PMID: 25654265

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Project description:Nonenzymatic RNA primer extension by activated mononucleotides has long served as a model for the study of prebiotic RNA copying. We have recently shown that the rate of primer extension is greatly enhanced by the formation of an imidazolium-bridged dinucleotide between the incoming monomer and a second, downstream activated monomer. However, the rate of primer extension is further enhanced if the downstream monomer is replaced by an activated oligonucleotide. Even an unactivated downstream oligonucleotide provides a modest enhancement in the rate of reaction of a primer with a single activated monomer. Here we study the mechanism of these effects through crystallographic studies of RNA complexes with the recently synthesized nonhydrolyzable substrate analog, guanosine 5'-(4-methylimidazolyl)-phosphonate (ICG). ICG mimics 2-methylimidazole activated guanosine-5'-phosphate (2-MeImpG), a commonly used substrate in nonenzymatic primer extension experiments. We present crystal structures of primer-template complexes with either one or two ICG residues bound downstream of a primer. In both cases, the aryl-phosphonate moiety of the ICG adjacent to the primer is disordered. To investigate the effect of a downstream oligonucleotide, we transcribed a short RNA oligonucleotide with either a 5'-ICG residue, a 5'-phosphate or a 5'-hydroxyl. We then determined crystal structures of primer-template complexes with a bound ICG monomer sandwiched between the primer and each of the three downstream oligonucleotides. Surprisingly, all three oligonucleotides rigidify the ICG monomer conformation and position it for attack by the primer 3'-hydroxyl. Furthermore, when GpppG, an analog of the imidazolium-bridged intermediate, is sandwiched between an upstream primer and a downstream helper oligonucleotide, or covalently linked to the 5'-end of the downstream oligonucleotide, the complex is better preorganized for primer extension than in the absence of a downstream oligonucleotide. Our results suggest that a downstream helper oligonucleotide contributes to the catalysis of primer extension by favoring a reactive conformation of the primer-template-intermediate complex.

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Replacing uridine with 2-thiouridine enhances the rate and fidelity of nonenzymatic RNA primer extension.

Publications

Replacing uridine with 2-thiouridine enhances the rate and fidelity of nonenzymatic RNA primer extension.

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